Bottom Line:
The solubility of F6'H was critical for the final yield.Umbelliferone and esculetin were also synthesized from glucose using engineered E. coli strains.The final yields of umbelliferone and esculetin were 66.1 and 61.4 mg/L, respectively.

Background: Coumarins are a major group of plant secondary metabolites that serves as defense compounds against pathogens. Although coumarins can be obtained from diverse plant sources, the use of microorganisms to synthesize them could be an alternative way to supply building blocks for the synthesis of diverse coumarin derivatives.

Results: Constructs harboring two genes, F6'H (encoding feruloyl CoA 6' hydroxylase) and 4CL (encoding 4-coumarate CoA:ligase), were manipulated to increase the productivity of coumarins. Escherichia coli expressing the two genes was cultured in medium supplemented with hydroxycinnamic acids (HCs) including p-coumaric acid, caffeic acid, and ferulic acid, resulting in the synthesis of the corresponding coumarins, umbelliferone, esculetin, and scopoletin. Cell concentration and initial substrate feeding concentration were optimized. In addition, umbelliferone, and esculetin were synthesized from glucose by using a ybgC deletion mutant and co-expressing tyrosine ammonia lyase and other genes involved in the tyrosine biosynthesis pathway.

Conclusions: To produce coumarin derivatives (umbelliferone, scopoletin, and esculetin) in E. coli, several constructs containing F6'H and 4CL were made, and their ability to synthesize coumarin derivatives was tested. The solubility of F6'H was critical for the final yield. After optimization, 82.9 mg/L of umbelliferone, 79.5 mg/L of scopoletin, and 52.3 mg/L of esculetin were biosynthesized from the corresponding HCs, respectively in E. coli. Umbelliferone and esculetin were also synthesized from glucose using engineered E. coli strains. The final yields of umbelliferone and esculetin were 66.1 and 61.4 mg/L, respectively.

Fig8: Production of umbelliferone (A) and esculetin (B) using B-CM11 and B-CM12 from glucose, respectively.

Mentions:
Esculetin was also synthesized from glucose by adding one additional gene (Sam5) that encodes a protein to convert p-coumaric acid into caffeic acid. We also tested the four constructs (pA-SeTAL, pA-aroG-SeTAL-tyrA, pA-aroGfbr-SeTAL-tyrAfbr, and pA-aroGfbr-ppsA-tktA-SeTAL-tyrAfbr) for the production of esculetin. The strain harboring pA-aorGfbr-SeTAL-tyrAfbr produced more esculetin than other strains harboring different constructs (data not shown). Therefore, B-CM12 was used for the production of esculetin. The product was confirmed based on HPLC retention time and the MS/MS fragmentation pattern (Figure 8 and data not shown). The optimal cell concentration was determined to be OD600 = 1.5, and the optimal incubation temperature was 25°C. Using the optimized conditions, production of esculetin was monitored. Esculetin production needs more time than umbelliferone because of an additional step for the conversion of p-coumaric acid into caffeic acid. Esculetin production continued to increase until 60 h, at which point approximately 61.4 mg/L esculetin was synthesized (Figure 7B), similar to the amount which was obtained from feeding caffeic acid. At 60 h, the production of esculetin was maximum and caffeic acid began to accumulate.

Fig8: Production of umbelliferone (A) and esculetin (B) using B-CM11 and B-CM12 from glucose, respectively.

Mentions:
Esculetin was also synthesized from glucose by adding one additional gene (Sam5) that encodes a protein to convert p-coumaric acid into caffeic acid. We also tested the four constructs (pA-SeTAL, pA-aroG-SeTAL-tyrA, pA-aroGfbr-SeTAL-tyrAfbr, and pA-aroGfbr-ppsA-tktA-SeTAL-tyrAfbr) for the production of esculetin. The strain harboring pA-aorGfbr-SeTAL-tyrAfbr produced more esculetin than other strains harboring different constructs (data not shown). Therefore, B-CM12 was used for the production of esculetin. The product was confirmed based on HPLC retention time and the MS/MS fragmentation pattern (Figure 8 and data not shown). The optimal cell concentration was determined to be OD600 = 1.5, and the optimal incubation temperature was 25°C. Using the optimized conditions, production of esculetin was monitored. Esculetin production needs more time than umbelliferone because of an additional step for the conversion of p-coumaric acid into caffeic acid. Esculetin production continued to increase until 60 h, at which point approximately 61.4 mg/L esculetin was synthesized (Figure 7B), similar to the amount which was obtained from feeding caffeic acid. At 60 h, the production of esculetin was maximum and caffeic acid began to accumulate.

Bottom Line:
The solubility of F6'H was critical for the final yield.Umbelliferone and esculetin were also synthesized from glucose using engineered E. coli strains.The final yields of umbelliferone and esculetin were 66.1 and 61.4 mg/L, respectively.

Background: Coumarins are a major group of plant secondary metabolites that serves as defense compounds against pathogens. Although coumarins can be obtained from diverse plant sources, the use of microorganisms to synthesize them could be an alternative way to supply building blocks for the synthesis of diverse coumarin derivatives.

Results: Constructs harboring two genes, F6'H (encoding feruloyl CoA 6' hydroxylase) and 4CL (encoding 4-coumarate CoA:ligase), were manipulated to increase the productivity of coumarins. Escherichia coli expressing the two genes was cultured in medium supplemented with hydroxycinnamic acids (HCs) including p-coumaric acid, caffeic acid, and ferulic acid, resulting in the synthesis of the corresponding coumarins, umbelliferone, esculetin, and scopoletin. Cell concentration and initial substrate feeding concentration were optimized. In addition, umbelliferone, and esculetin were synthesized from glucose by using a ybgC deletion mutant and co-expressing tyrosine ammonia lyase and other genes involved in the tyrosine biosynthesis pathway.

Conclusions: To produce coumarin derivatives (umbelliferone, scopoletin, and esculetin) in E. coli, several constructs containing F6'H and 4CL were made, and their ability to synthesize coumarin derivatives was tested. The solubility of F6'H was critical for the final yield. After optimization, 82.9 mg/L of umbelliferone, 79.5 mg/L of scopoletin, and 52.3 mg/L of esculetin were biosynthesized from the corresponding HCs, respectively in E. coli. Umbelliferone and esculetin were also synthesized from glucose using engineered E. coli strains. The final yields of umbelliferone and esculetin were 66.1 and 61.4 mg/L, respectively.